Describe the role of bioreactors in eliminating pollutants?

What are bioreactors?

A bioreactor is a large tank where biological degradation of contaminants is isolated and

controlled following series of biological reactions under sterile and controlled environmental
conditions.

Bioreactor degrades contaminants in groundwater and soil in the presence of microorganisms. In

contrast to attenuation and in-situ bioremediation, Bioreactors control and optimize issues of
ineffective indigenous microorganisms or low indigenous microbial populations.

In Bioreactors microbial processes are optimized related to contaminated media and nature of
pollutants. Bioreactors provide a controlled environment to control critical process parameters to
optimize the microbial bioremediation process. There is also flexibility in size and configuration
of bioreactors. Bioremediation in bioreactors involves excavation of soils/sediment,
transportation and controlled handling of the contaminated media. Pretreatment of contaminated
media i.e. drying and crushing are also required.

Bioremediation using microbial bioreactors finds application in soil, air and water environments
including:

Waste water and industrial effluent treatment

Microorganisms are the primary agents of any biological wastewater treatment. Microorganisms
are already present in waste water systems and feed on complex substances in the wastewater
converting them to simpler substances thus assisting in achieving the treatment. Trickling filters,
membrane bioreactors, slurry phase reactors and upflow anaerobic sludge blanket bioreactors
(UASB) are some of the reactors that are used in waste water and industrial effluent treatment.

Soil and land treatment

Contaminants successfully treated include diesel fuel, fuel oils, oily sludge, wood-preserving
wastes (PCP, PAHs, and creosote), coke wastes, and certain pesticides [6, 8, 9]. Soil
bioremediation has proven most successful in treating petroleum hydrocarbons and other less
volatile, biodegradable contaminants. Slurry phase, stirred tanks, biofilters, partitioning phase
and packed microbial reactors find application in contaminated soil remediation.

Control of air pollution

Microorganisms are used in the bioremediation of organic and inorganic air pollutants in spent
gases before release or escape into the atmosphere [5, 9]. Microorganisms oxidize pollutants
such as H2S, SO2, VOCs, and reduce pollutants such as NOx to nitrate and this assist to prevent
likely environmental, health hazards and nuisances [5]. Bioscrubbers and biofilters are some of
the bioreactor types often used in control of air pollution.

Solid waste management

Microorganisms are chiefly responsible for the biodegradation of organic wastes in nature and
they drive the decomposition processes that occur in landfills and composts. Anaerobic digesters
are often applied mostly in the biotreatment solid waste.

Factors affecting microbial bioreactor performance

A number of issues are at play in all bioremediation technologies including when bioreactors are
used. These are those that concern the contaminant, microbial community and the design,
optimization and monitoring of the process [6, 8, 9]. The microbial science of bioremediation is
therefore approached from many scientific frontiers: abiotic interactions (solubility, transport,
sorption and photolysis), biotic interactions (taxonomic diversity, physiological, genetic and
ecological interactions). In the design and operation of bioreactors in remediation, many of these
factors have to be optimized and controlled for best reactor performance [5, 10, 11, 12].

Variables that affect the operation and efficiency of a microbial bioreactor relate to biotic and
abiotic factors that affect microbial growth and those factors that relate to the reactor design and
configuration. Factors that affect microbial growth and activities in bioreactors include;
environmental factors (temperature, pH, moisture), pollutant mix, pollutant concentration,
macronutrient [5, 10, 11, 12]. Factors on reactor design include; size, configuration and mode of
operation.

Environmental related factors

Environmental conditions (temperature, pH, oxygen availability/electron, and salinity) affect
growth; the metabolic activities of microorganisms and to some extent the behavior of the
pollutant such as solubility and volatility [11]. In any process optimization for biodegradation, it
is always necessary to investigate the effects of the environmental conditions and optimize the
process in relationship to all the relevant environmental conditions. Tekere et al. [13], established
the optimum growth conditions with respect to pH, aeration and nutrients in the growth and
degradation of pollutants by white rot fungi and found that optimized conditions result in high
enzyme and degradation activities.

Temperature

There is always a temperature range at which microorganisms grow and survive (i.e., minimum,
optimum and maximum survival temperature). In addition, there is always a temperature
optimum at which biochemical processes take place to achieve required bio treatment by each
microorganism [13]. Extremes of temperature (too low or too high) affect both microbial growth
and microbial enzyme catalyzed reactions [2]. With an increase in temperature within
appropriate range, microbial metabolism increases and thus the rate of the bioremediation
processes.

Increased temperatures lead to higher solubility of many chemicals, and increased fluidity and
diffusion rates. For example with pollutants, such as PAHs and heavy metals, their solubility and
in turn bioavailability increases with temperature [2, 7]. Temperature is thus a critical factor in
the optimum operating efficiency of bioreactors to achieve best biotreatment results. Often
specialized bioreactors are designed with provision for temperature control.

pH

Similar to temperature, pH affects microbial growth and metabolic processes. pH influences

microbial cell ionic properties thus microbial growth. Microorganisms have minimum, optimum
and maximum pH of growth with most bacteria for example growing optimally at pH 6–7.5,
though there are some which thrive best at acidic pHs (acidophiles) or at alkaline pH
(alkaliphiles). Fungi generally grow at pHs lower than that of bacteria. Reactor operating pH has
to be set to provide the best pH conditions for growth and enzyme activities. Behavior of
pollutants is also influenced by pH thus affecting their bioremediation. For example with metals,
pH affects the redox and solubility of metals, different forms and valence have different effects
on microorganisms [14]. Metal solubility increases with a decrease in medium pH and alkaline
pH favor metal ion precipitation. Often lower pH values are required for metal attachment to the
microbial cell surface [7, 14]. Microorganisms that produce acids result in increased solubility of
the metal ions [10]. To provide for best pH conditions, buffers are used in media formulations,
acids and bases can be added during the bioreactor process [13].

Nutrients

Nutrients are required for growth and metabolism of the microorganisms. Several elements are
required in biosynthesis and energy production. Carbon is the most basic element of living forms
and is needed in greater quantities than other elements. Other elements that are important in
ensuring a balanced nutritional bioreactor environment depending on the type of microorganism
include hydrogen, oxygen, nitrogen, sulfur, phosphorus, iron, calcium and magnesium [10, 11].
All necessary macro- and micro- nutrients requirements are provided in reactor media.
Microorganisms can use the pollutants they are degrading as primary energy sources or a
primary source of energy is provided to the microorganism in the case of co metabolism of the
pollutants.

Moisture

Water is required to support microbial growth and catalysis. Cellular chemical reactions occur in
aqueous conditions and water is required to ensure the correct osmotic pressure is maintained for
microbial growth. The amount of water available for microbial growth is called (aW). Most
microorganisms grow at water activities of 0.98 or higher, osmotolerant species can however
grow at a range of low aW [11].

Electron acceptors

The presence of electron acceptors, e.g., oxygen in aerobic microbes and NO31−, SO42− and Fe
(III) oxides in case of anaerobic microbes, also affects the biodegradation processes.

Reactor design related factors

Bioreactors have to provide for the best conditions for microbial growth and biochemical process
to occur. The reactor size, configuration and mode of operation are key reactor design factors.
The reactor should provide favorable physical, biological and the combined physical-chemical
conditions for the best biological remediation processes to be achieved. In designing the
bioreactor, favorable physical conditions for transport of gases and liquids and solids over time
that ensure that the physical entity of the bioreactor is favorably adapted to the biological system
that performs the bioreactions are required [12, 15]. On the other hand there is need to ensure
that the biophysical and biochemical events taking operate at optimum levels under real situation
application.

Polluted samples for remediation can be fed into the reactor either as dry or slurry matter [9].
Pollutants with hydrophobic properties are often unavailable for microbial degradation,
particularly if they are bound to soil matrix [7]. Their degradation is therefore limited by their
transfer to liquid [4]. Minimizing mass transfer resistance was found to be a key factor in the
degradation of hexachlorocyclohexane (HCH) in slurry batch bioreactors [4].

Despite the rapid development of bioreactors due to their widespread use in biotechnology, the
aspects of maintaining stability and rates of bioprocesses are still areas to be addressed. Poor
bioreactor construction and design, leading to inadequate mixing, may jeopardize the stability
and performance of the process [15]. Mixing prevents thermal stratification, help maintain
uniform conditions in the reactor, ensure good contact between microbial culture and media
reactants. The importance of mixing in bioreactor cannot be over emphasized, poor mixing affect
microbial process efficiency.

Hydraulic retention times (HRT) required to achieve the necessary remediation goals in the
bioreactor have to be determined and optimized. Longer HRTs result in poor substrate loading
which diminishes the microbial population, whereas shorter ones do not allow microorganisms to
effectively degrade the pollutant and can result in microbial wash out from the system [16].

Organism related factors

Organism related factors include population density, composition, inter and intraspecific
interaction. Microbes are the most diverse forms of life and have developed a wide range of
metabolic pathways that enable them to cope under the varying ecological conditions including
exposure to xenobiotics. A whole range of environments ranging from aerobic, anaerobic, acidic,
alkaline, and low to high temperature have been utilized as sources of microorganisms for
bioremediation [13]. Only certain species of bacteria and fungi have proven their ability as
potent pollutant degraders [13]. In the natural environment degradation of pollutants is often
achieved through complex microbial population interactions. Single or mixed microbial cultures
are used for pollutant remediation in bioreactors. In the event where bioagumentation is applied
the introduced organisms need to be able to co-exist with indigenous residents.

Different microorganisms often have different metabolic capabilities, to this extend the
evaluation of several strains of different microbial players have to be investigated in order to
come up with the best degraders [13]. In screening and comparison of the bio-degradation of
PAHs by white rot fungi [17], found out that newly screened white rot fungi strains had higher or
comparable degradation capacity to the model well applauded P. chrysosporium, and these
strains did not accumulate the metabolite quinone which accumulates as a dead end metabolite
in P. chrysosporium.

Polluted environments provide sources of microorganisms resistant or acclimatized to the

pollutant [18]. However microorganisms that are known to have certain inherent physiological
characteristic, e.g., metabolism of known substrate with structural similarity to xenobiotics of
interest and/or adaptation to certain environmental conditions can be selected. This is the case in
several studies that used microorganisms for pollutant degradation [11, 17, 18, 19].

Pollutant related factors

Factors that affect bioremediation in bioreactors that are related to the pollutant include: nature
of pollutant, i.e., the physical and chemical properties including solubility, volatility, molecular
complexity, concentration and toxicity. Investigations for most pollutant biodegradation have
centered on how different concentrations, mixed pollutants, solubility and molecular structure
can affect microbial bioremediation [17, 20]. In the case of PAHs, degradation decreases in the
order alkane> branched chain alkanes>low molecular weight aromatics> cycloalkanes [17]. It
should be noted however that some pollutants are resistant to biodegradation (recalcitrant, i.e.,
resistant to degradation) they are degraded at very low pace even if the right microbial
population and conditions are present.
. Microbial bioreactors in bioremediation

Several laboratory, and pilot bioremediation studies have been done using microbial (fungi and
bacteria) bioreactors [6, 8, 17, 18, 20]. Bioreactor technologies may offer effective means for
treatment of many contaminants in groundwater, soil and air [4, 5, 7, 12]. The bioreactor type of
choice for any application should be easy to operate and maintain for the selected purpose and
application. Table 1 presents some of the studies that involved the use of bioreactors in
bioremediation. Flexibility to design bioreactor tailor made for different processes and
remediation applications makes the use of bioreactors in bioremediation attractive [9]. The
design should accommodate high biomass from cell growth, supply of necessary nutrients and
also removal of waste components from the system. A description of some bioreactor types and
their application is given in Sections 3.1–3.7.

3.1 Slurry phase bioreactors

Slurry phase bioreactors, as the name implies treats polluted media that is within a slurry phase.
Alternative names are bio-slurry reactors and slurry phase biological treatment. Slurry
bioreactors offer an ex situ environmentally friendly way for remediating mostly soils and
sediments from petrochemical hydrocarbons, tars, creosotes, chlorinated solvents, herbicides,
pesticides and explosives or when a solid substrate that is formulated into a slurry is used
[4, 6, 25, 26]. Hydrophobic nature of most persistent chemicals makes them sorb to soil or
sediments and not easily accessible for biodegradation.

Operation of the slurry reactor can be in batch, semi-continuous and continuous mode, with the
batch process being the most common one [6, 26]. Figure 1 shows an illustration of a simplified
slurry reactor. Water is mixed with the contaminated solid matrix in suitable ratios and this
enhances contact between microorganisms, pollutant, media and oxygen. Pollutants that are
solubilized become more bioavailable. Table 2 shows some of the studies that have involved the
use of slurry phase bioreactors in bioremediation.

3.2 Partitioning bioreactors

Partitioning bioreactors are used in bioremediation when two phases need to be achieved, e.g.,
such as for organic solvents or water immiscible compounds in aqueous solutions. Reactors are
designed with the aqueous and organic phase, and can be single or multiphased [24]. With toxic
hazardous waste, toxicity to degrading microorganisms is a problem. In partitioning bioreactors,
there is a two-phase system where a water immiscible and biocompatible organic solvent is
allowed to float on the surface of a cell containing aqueous phase [45]. This means that high
amounts of hazardous waste dissolved in a solvent can be added to the reactor without the
microorganism experiencing inhibitory concentrations of the pollutant [24, 45, 46]. A rigorous
process involving selection of the solvent, taking into consideration the biological, physical,
operational, environmental and economic factors is necessary in developing an efficient
partitioning biotreatment system. Partitioning reactors find application in the remediation of
toxic compounds from petrochemical industry such as benzene as well as VOC in waste gases of
many industrial processes [45, 47, 48]. Angelucci et al. [49], successfully tested a continuous
two-phase-partitioning reactor in the treatment of tannery wastewater. Several other studies
involving phase partitioning bioreactors are described [24, 45, 46, 47, 48, 49, 50].

3.3 Stirred tank bioreactors

A continuous stirred tank bioreactor consists of a cylindrical vessel with motor driven central
shaft that supports one or more agitators (impellers). Stirred tank bioreactors are the
predominantly used design for submerged cultures. Stirred tank bioreactors are mechanically
agitated where the stirrers are the main gas-dispersing tools and provide high values of mass
transfer rates coupled with excellent mixing. Advantages of the STR include the efficient gas
transfer to growing cells, good mixing of the contents and flexible operating conditions, besides
the commercial availability of the bioreactors. The main shortcoming of the stirred tank
bioreactor is its mechanical agitation which requires energy and stirring can cause shear strain on
microbial cells.

Gargouri et al. [7] evaluated the use of a continuously stirred tank bioreactor (CSTR) in the
treatment of hydrocarbon-rich industrial wastewaters and achieved successful bioremediation
using an acclimatized microbial consortium. The residual total petroleum hydrocarbon (TPH)
decreased from 320 –8 mg TPH l−1. The reactor used is shown in Figure 2. Bi [51], applied a
continuously stirred tank reactor for bioremediation of ethanol, toluene and benzyl alcohol by P.
putida.

3.4 Biofilters
A basic biofilter bioreactor consist of a large media bed where pollutants are passed through and
get degraded by the microorganisms. Biofilters are amongst the oldest environmental
bioremediation techniques. Biofilters are used mostly in waste water treatment as well as in the
control of air pollution [34, 52, 53]. A number of materials are used for bed media such as peat,
composted yard waste, bark, coarse soil, gravel or plastic shapes. A typical example of a biofilter
is the trickling filter which finds extensive application in the treatment of different liquid
effluents or waste waters or waste that is constituted into liquid. A trickling filter is usually a
round, vertical tank that contains a support rack and is filled with aggregate, ceramic or plastic
media and in the middle of the tank is a vertical pipe that has a rotary connection with spray
nozzles on the top end [34]. A spray arm is attached to the rotary connection and has spray
nozzles installed along its length for distribution of the waste water. Microorganisms grow in
biofilm forms on the packing material surface and are responsible for the degradation of the
pollutants from the effluent. Schmidt and Anderson [34] described the use of a trickling biofilter
in the removal of high concentrations of 1-butanol from contaminated air. The potential
application of the biotrickling filter in industrial off gas treatment was evaluated in the removal
of high concentrations of 1-butanol from contaminated air with efficiency exceeding 80% for
butanol concentrations of 0.4–1.2 g m−3 [34]. The laboratory-scale perlite-packed biotrickling
filter was operated for 60 days and demonstrated effective and efficient removal of butanol
concentrations up to 4.65 g m−3 with a maximum elimination capacity of 100 g m−3 h−1 [34].

3.5 Packed bed bioreactors

Packed bed bioreactor systems provide for microbial growth on fixed film substrata. In order to
obtain compact reactors and ensure greater treatment reliability, fixed film reactors are used.
They offer the advantage that dilute aqueous solutions can be remediated at high biomass
without the need to separate biomass and the treated effluent [13, 54]. In packed bed biofilm
biotreatment processes, unlike suspension cultures there is no need to incorporate special
measures such as centrifugation and membrane filters to retain the biomass. This feature makes
the use of packed bed reactors particularly appropriate in bioreactors systems where large
substrate—flow through is required. The concentration of cells in a given volume may be
increased, a factor that leads to enhanced efficiency/productivity of the bioreactor and decreased
volume of bioreactors [55]. While high biomass concentrations can be easily maintained, the
medium to biofilm mass transfer of substrate is the rate limiting process in packed bed
bioreactors [54, 56]. Within the biofilm there are considerable differences in the
microorganisms’ microenvironment, depending on the distance from the surface of the biofilm
[54]. Substrates such as oxygen, carbon and nitrogen sources have to cross the biofilm—liquid
interface by diffusion, thus a diffusion gradient occurs. To calculate the kinetics of conversion in
the biofilm processes, two important processes that occur in the system are considered and these
are (i) transport of solutes over the biofilm and (ii) combined reactions and diffusion inside the
biofilm [54]. In the packed bed reactors, development of excess microbial biomass also occurs
leading to hydraulic channeling or loss of interstitial fluid volume. To overcome the severe
constraints of hydraulic hold up within the interior of the reactor extra-capillary space transverse
flow bioreactors were developed [57].

Selection of suitable substances as packing materials is an important consideration. Materials

that have been used include nylon web, polyurethane foam, silicone tubing, sintered glass,
porous ceramics, propylene, stainless steel, agarose and agar gel beads
[58, 59, 60, 61, 62, 63, 64, 65, 66, 67]. The ideal support should be chemically inert in
physiological growth medium, rigid and porous to facilitate mycelial attachment and re-usable
after removal of the fungus. Figure 4 shows a Simplified diagram of a laboratory based packed
bed bioreactor. Examples of remediation studies in packed bed reactors are given in Table 3.

3.6 Airlift bioreactors

Airlift bioreactors can provide an attractive treatment alternative for treatment of gaseous or
volatile air pollutants. Frequently, the most limiting factor in the performance of these reactors is
that they are susceptible to being limited by gas-liquid mass transfer and by poor mixing of the
liquid phase, particularly when they are operating at high cell densities [68, 69]. The bioreactor
performance is dependent on the pumping (injection) of air and the liquid circulation. The airlift
bioreactor can have a forced flow in an internal or external loop as shown in Figure 5. Specific
volatile organic chemicals may be completely degraded by a microorganism at normal
temperature and pressure without producing a second polluted byproduct [70]. Nikakhtari and
Hill [68], applied and External Loop Airlift Bioreactor with a small amount (99% porosity) of a
stainless steel mesh packing inserted in the riser section for bioremediation of a phenol polluted
air stream. Phenol removal of 100% was achieved using the bacterium Pseudomonas putida, and
at a phenol loading rate of 22,160 mg h−1 m−3, thus demonstrating the novelty and potential
VOCs bioremediation application of the reactor at high loading rates. Figure 5 presents a
schematic diagram of airlift bioreactor. Several other studies involving the use of airlift
bioreactors [19, 69, 70, 71].

3.7 Membrane bioreactor

Membrane bioreactors (MBR) combine the use of a membrane that forms a filtration system and
the biological process. The membrane provides a physical barrier that separates the liquid from
the solid and ensures retention of the solids and good quality effluent. The quality of the treated
effluent from the membrane bioreactor is of high quality than that achieved by employing other
techniques, enabling optimal functioning of the secondary treatment system [72, 73]. MBR offer
the advantages that often smaller tank size is used and filtration function of the membrane
ensures that solids are separated from treated effluent. Membrane fouling has been recognized
however as a major drawback in the application of membrane bioreactors in bioremediation.
Also membranes are often expensive thus making the process costly. Development of low cost
membrane filters is an ongoing feature in the science of MBR [72]. MBR reactors have been
used in the biological treatment of domestic and industrial waste water. MBR have been
evaluated in the remediation of pentachlorophenol in concentration ranges that occur in waste
water [73], textile waste water [27], 1,2-dichloroethane, 1,2-dichlorobenzene and 2-chlorophenol
[30].

3.8 Other bioreactors in bioremediation

Due to flexibility in bioreactor designs, the configuration of reactors is numerous. While an

effort has been made here to describe some of the common bioreactors used for different
bioremediation applications, several other bioreactor types have not been discussed. These
include the UASB which find major application in anaerobic digestion of waste waters as well as
solid wastes, bio-scrubbers which are applied in off gas air pollution control, continuous stirred
tank reactors as well as rotating contactor reactors.

It is evident that a wide range of microbial bioreactors have been developed and evaluated in the
bioremediation of a wide range of pollutants in water, air and soil. Also a wide range of
pollutants in physical and chemical properties are amenable to microbial degradation. Very
diverse microbial species have the capability of pollutant degradation naturally and the use of
well-developed optimized microbial bioreactors ensure improved rates of degradation when
compared to degradation that happens in situ in the environment under natural environmental
conditions.